Protein ubiquitination is a reversible post-translational modification conserved in all eukaryotes. Whereas the attachment of ubiquitin (Ub) serves as a signal in diverse processes, deubiquitinating enzymes (DUBs) regulate these processes by removing the Ub signal. The human genome encodes ~95 DUBs. How their specificities and enzymatic activities are regulated has only recently begun to be addressed. We have shown that Uch37, a DUB conserved from fission yeast to man, is partitioned between two structurally and functionally distinct multiprotein complexes, the proteasome regulatory particle (RP) and the human Ino80 chromatin- remodeling complex (hINO80), and that these complexes have opposing effects on Uch37 deubiquitination activity. This proposal aims to understand mechanistically how Uch37 activity is regulated by its associated proteins; it also will investigate the functions of Uch37 in the two complexes. In Aim 1, we will elucidate how Uch37-associated proteins modulate its activity and substrate specificity. Structural and functional analyses of the Uch37-Rpn13 complex will reveal how Uch37 is activated by RP subunit Rpn13 and how it can process polyUb specifically from the distal end of the chain. By reconstituting a RP subcomplex, we will identify RP subunits whose interactions with Uch37 enable polyUb disassembly activity. We will also develop Uch37 mutants that specifically interact with either the RP or hINO80. These mutants will be used to resolve functions of Uch37 associated with either complex. In Aim 2, we will analyze the functions of Uch37 in substrate processing by the proteasome and in chromatin remodeling by hINO80. We will elucidate how Uch37 and Rpn13, a DUB-Ub receptor duo, function together in vivo, and we will establish an in vitro system to understand how Uch37 assists degradation in a substrate-specific manner. Finally, we will define Uch37's role in regulating histone ubiquitination levels and how this function affects chromatin remodeling by hINO80. These studies will dramatically enhance our understanding of the regulatory function of deubiquitination in both proteolytic and non-proteolytic processes.